Contractile function in ventricles surviving substantial myocardial infarction (MI) is improved by exercise training. In the 1st grant period, we showed that a program of high intensity sprint training (HIST) instituted shortly after MI was effective in reversing many abnormalities in myocyte excitation-contraction coupling. In the current grant period, we focused on the cardiac Na+/Ca2+ exchanger (NCX1) and sarco(endo)plasmic reticulum Ca-ATPase (SERCA2) as targets by which HIST mediated its beneficial effects. We demonstrated that NCX1 plays a major role in the myocyte contractile and [Ca2+]i transient abnormalities observed post-MI, and that HIST improved myocyte contractile function at least partly by enhancing NCX1 activity. By contrast, augmenting SERCA2 activity post-MI only partially ameliorated contractile dysfunction post-MI. We also demonstrated that phospholemman (PLM), a novel endogenous inhibitor of NCX1, was increased post-MI. In this competitive renewal, we wish to focus on the role of NCX1 and PLM on post-MI cardiac dysfunction, using genetically engineered mouse models. We Hypothesize: (i) increased inhibition of NCX1, either by PLM overexpression or increased PLM phosphorylation, accounts for contractile dysfunction post-MI; (ii) the beneficial effects of HIST are largely mediated by enhancement in NCX1 function. We will use PLM-knockout (KO), conditional NCX1-KO, and transgenic mice with controlled NCX1 expression (tet-off) to test our hypotheses. Our Specific Aims are: (1) a): to enhance NCX1 function by knocking out PLM, this should improve post-MI contractility and calcium homeostasis; b): to decrease NCX1 function by re-introducing PLM in PLM-KO mice by recombinant adeno-associated virus (rAAV) delivery, this should negate the positive effects of PLM-KO on post-MI myocyte contractility; c) differentiate the effects of PLM on NCX1 and the Na+ pump by using PLM serine mutants; d) exercise train both WT and PLM-KO mice post-MI and evaluate the effects on myocyte contractility, NCX1 function and PLM expression/phosphorylation. (2) a): to enhance NCX1 function by overexpressing NCX1 using rAAV6 delivery 3 weeks before MI, this should result in enhanced myocyte function; b): turn on NCX1 expression in a tet-off transgenic mouse expressing NCX1 during the peri-infarct period, this should improve myocyte contractility; c): downregulate NCX1 function by r-AAV6 mediated anti- sense delivery; or eliminating NCX1 expression by creating a conditional cardiac-specific NCX1 KO (MerCreMer, NCX1 floxed) mouse, this should result in reduced contractility post-MI; and d) evaluate the effects of HIST on WT and NCX1 KO mice post-MI on myocyte contractility. The critical aspect of the grant proposal is to systematically evaluate the role of NCX1 and PLM on cardiac function post-MI, not only at the cellular level, but also at the whole animal level so that long-term survival can be evaluated. Understanding the role of NCX1 and PLM and their regulation post-MI will allow for novel therapies for ischemic cardiomyopathy. PUBLIC HEALTH RELEVANCE: Project Narrative Myocyte contractile dysfunction significantly contributes to heart failure. This proposal uses 3 different genetically altered mice specifically engineered to critically and unequivocally evaluate the role of sodium- calcium exchanger in heart failure after a heart attack and the beneficial effects of exercise after infarction.